More than 80 types of human papillomavirus (HPV) have been described, many of which are associated with a wide variety of biological phenotypes, from benign proliferative warts to malignant carcinomas (for review, see McMurray et al., Int. J. Exp. Pathol. 82(1): 15–33 (2001)). HPV6 and HPV11 are the types most commonly associated with benign warts, whereas HPV16 and HPV18 are the high-risk types most frequently associated with malignant lesions. More than 90% of cervical carcinomas are associated with infections of HPV16, HPV18 or the less prevalent oncogenic types HPV31, -33, -45, -52 and -58 (Schiffman et al., J. Natl. Cancer Inst. 85(12): 958–64 (1993)). The observation that HPV DNA is detected in more than 90% of cervical cancers provides strong epidemiological evidence that HPVs cause cervical carcinoma (see Bosch et al., J. Natl. Cancer Inst. 87(11): 796–802 (1995)).
An effective vaccine against HPV is needed to prevent the development of cervical dysplasias and carcinomas and their associated morbidity and mortality. Vaccines to both the low risk and high risk HPV types are currently being tested in clinical trials. The ability to determine the presence of HPV type-specific antibodies to several HPV types in clinical samples would be useful for both monitoring the efficacy of prospective vaccines and for natural history infection studies.
Several different methods have been developed to quantify neutralizing antibodies to HPVs, including in vivo neutralization assays, in vitro pseudo-neutralization assays, competitive radioimmunoassays (cRIAs), and enzyme-linked immunosorbent assays (ELISAs). However, each of these techniques possesses one or more limitations that preclude testing large numbers of patient sera for use in large vaccine clinical trials and natural history studies.
The most common technique for measuring neutralizing HPV antibody titers is the athymic mouse xenograft system (Bryan et al., J. Med. Virol. 53(3): 185–88 (1997); Kreider et al., Nature 317(6038): 639–41 (1998). In this assay, sera from individuals is mixed with infectious HPV and added to foreskin tissue, which is then implanted under the renal capsule of athymic mice. The implants are monitored for histological changes and for HPV DNA by in situ hybridization. Because such histological changes can take several months to develop, this method is unsuitable for high throughput analysis.
Due to the technical difficulties in testing a large number of sera in the athymic mouse xenograft system, several complementary assays have been developed to measure neutralizing and non-neutralizing antibody titers. These include in vitro pseudo-neutralization assays (Bryan et al., supra; Roden et al., J. Virol. 70(9): 5875–83 (1996)), competitive RIAs (Palker et al., Vaccine 19(27): 3733–43 (2001)) and virus like particle (VLP)-based ELISAs (Wideroff et al., J. Infect. Dis. 172(6): 1425–30 (1995)). Studies have shown that the pseudo-neutralization assay can be 20- to 30-fold less sensitive than HPV-VLP ELISAs (Nardelli-Haefliger et al., J. Virol., 73(11): 9609–13 (1999) and up to a 100-fold less sensitive than a competitive RIA (Palker et al., supra). However, because ELISA-based assays can only detect one analyte at a time, this technique is impractical for analyzing large numbers of clinical samples where detecting and quantifying antibody titre to several HPV types is desirable.
Recently, methods have been developed to simultaneously measure multiple analytes using particle-based flow cytometric assays. This approach has been used to measure numerous types of soluble analytes, including antibodies specific to various antigens, and to quantify the cell surface expression of various receptors (for review, see Vignali, J. Immunol. Methods 243: 243–255 (2000)). Schlottman and colleagues used this method in pneumococcal vaccine studies to measure the antibody response to a pneumoconjugate vaccine (Martins, Clin Diagn Lab Immnwol: 9(1): 41–45 (2002)). Bellisario and colleagues developed a particle-based flow cytometric assay to simultaneously measure antibodies to HIV-1 antigens p24, gp160 and gp120 in newborn dried blood-spot specimens (Early Human Devel. 64: 21–25 (2001)). Both of these studies utilized simple capture-type assays where the desired analytes are directly detected in a sample.
Despite the development of the immunoassays described above, it would be advantageous to develop an assay to measure HPV type-specific antibodies to neutralizing epitopes that is highly sensitive and reproducible, and that requires reduced man-hours compared to methods disclosed in the art. An immunoassay that measures HPV type-specific antibodies to several HPV types simultaneously would be preferable to a single serological assay, which requires serial experimentation.